Calaxin is a key factor for calcium-dependent waveform control in zebrafish sperm

This study demonstrates that Calaxin’s calcium-binding activity is necessary for the calcium-induced asymmetric beating of cilia but dispensable to stabilize outer arm dynein.

Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authorsWe encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript.If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information.These files will be linked online as supplementary "Source Data" files.***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available.Failure to provide original images upon request will result in unavoidable delays in publication.Please ensure that you have access to all original microscopy and blot data images before submitting your revision.***- --------------------------------------------------------------------------Reviewer #1 (Comments to the Authors (Required)): The authors demonstrate two distinct functions of calaxin using zebrafish: Ca2+-dependent regulation of flagellar asymmetry in sperm and Ca2+-independent stabilization of KV cilia.The experiments are all well designed and performed and thus the paper greatly contributes in understanding the function of calaxin in ciliary motility.I would simply like to recommend the authors to address following issues before publication.
1.It is difficult to determine the sperm head axis in teleost sperm with round heads.The authors thus employed the tangent angle in zebrafish sperm in flagellar analysis.This is reasonable and the results well describe the difference in wave propagation and asymmetry between WT and calaxin -/-sperm.The abnormal wave propagation at the tip region is also obvious in calaxin -/-sperm.The authors showed the sperm flagellar asymmetry by basal curvature.This way may indirectly describe the asymmetric waves but does not directly express the asymmetric properties between the principal and reverse bend.The authors may add the "asymmetry index", which is the ratio of maximal curvatures of both bends (Mizuno et al, 2012).
2. In zebrafish the demembranated sperm are not fully reactivated under high concentration ATP (>0.2mM).However, many studies show that OAD functions in increasing the beat frequency of a flagellum under high concentration of ATP.Data under high concentration ATP should strengthen this paper.
3. It is interesting that the calaxin KO affects OAD stability, notably observed in the tip or distal region of sperm flagella (Figure 1, immunofluorescence).Why is OADs in the proximal most region normally assembled without calaxin and only a limited distal region affected?4. The kymographs in Fig. 2C show the independency of Ca2+ binding to calaxin in the motility of KV cilia and the zebrafish body laterality.Beat frequency of calaxin -/-flagella shows lower than those of WT and E130A mRNA injected embryos.However, the irregularly vibrating movement is observed in calaxin -/-zebrafish and also in calaxin -/-sperm.Is this due to the lack of calaxin function in bend propagation? 5. Figure 3A: The caption for the colors could not been linked to the immunofluorescence, instead to the tangent angle plot.The authors had better to layout the position of caption and immunofluorescent image.
6. Line 177: 50 μM ATP 50 μM ADP condition; may be better to insert "plus" or "-" between ATP and ADP?This also appears in other places in the text and figure.This manuscript describes an experimental study of the role of Calaxin in stabilizing outer dynein arms and influencing asymmetry of the flagellar waveform of Zebrefish sperm.Using a transgenic mutant with in which Calaxin is deficient in calciumbinding, outer dynein arms are stabilized but calcium-induced waveform asymmetry is not recapitulated, the authors show that Calaxin is a calcium-dependent regulator of dynein but calcium independent stabilizer of outer arm dynein.The studies are well designed and the data support the conclusions.
My request to the authors is to clarify and provide more details on the waveform analysis described briefly in lines 198-202."Instead, we selected sperm with their heads fixed to the glass slide and freely swimming flagella and calculated the tangent angle relative to the recorded frame.To obtain the time-and spatial average, we plotted tangent angles from ≧5 traces over one beat cycle followed by the fitting with the linear equation.Basal curvature was defined as the slope of the fitted equation."
"We also calculated the asymmetry index as in Ciona sperm (Mizuno et al., 2012) The result showed a similar trend as the basal curvature: the pCa4 condition led to higher asymmetry indices, with Tg E130A exhibiting a weaker elevation compared to WT and Tg WT.
In the EGTA condition, the reactivation rate was better, but higher ATP concentration gradually decreased it.Lower reactivation rate a) made the data collection more difficult, because only a small portion of reactivated sperm had freely swimming tails (demembranated flagella easily adhered to the glass slide), and b) raised the possibility that the sperm flagella is not properly reactivated.Thus, we decided to use 50 μM ATP + 50 μM ADP condition, where more than half of sperm were reactivated under the presence of calcium or EGTA.
To clarify these points, we modified lines 175-181 as follows. (before) Comparison of different ATP and ADP concentrations showed that higher ATP concentration induced faster beating frequency and that the supplementation of ADP reduced the variation of beating frequencies.We chose 50 μM ATP 50 μM ADP condition that induced relatively slow, uniform beating, which made observation easier (Fig. S2 A).

(after)
Comparison of different ATP and ADP concentrations showed that higher ATP concentration induced faster beating frequency and that the supplementation of ADP reduced the variation of beating frequencies (Fig S2A).On the other hand, higher ATP concentration resulted in lower reactivation rate especially in the pCa4 condition (Fig S2B).Thus, we chose 50 μM + ATP 50 μM ADP condition to enable the stable beating frequency and the higher reactivation rate.
To discuss this point, we added the following sentences in lines 285-291: "Intriguingly, we have previously shown that OADs are also reduced in the proximal region of calaxin -/-flagella (Yamaguchi et al., 2023).This indicates that Calaxin knockout destabilizes OADs in any region, and some other factors may accelerate the phenotype in the distal region.
Thank you for the comment.We moved the caption below the immunofluorescent image.
The studies are well designed and the data support the conclusions.

-Provide more details on the waveform analysis in Methods or Supplementary Material
We modified Supplementary Figure 2E and added "Waveform analysis" paragraph in the Materials and Methods section.Python programs we used are now available on GitHub (https://github.com/motohiromorikawa/Calaxin-analysis).Thank you for submitting your revised manuscript entitled "Calaxin is a key factor for calcium-dependent waveform control in zebrafish sperm".We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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7.
Line 200: "relative to the recorded frame" is unclear.Is it the tangent angle along a flagellum in each frame recorded?Reviewer #2 (Comments to the Authors (Required)):

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Dear Dr. Eric Sawyer, Executive Editor, Life Science Alliance It is our pleasure to submit the revised version of our manuscript: "Calaxin is a key factor for calcium-dependent waveform control in zebrafish sperm", to Life Science Alliance.Compared to the original submitted version, we have improved the descriptions and analyses, thanks to the comments and suggestions by reviewers.Specific comments and answers are summarized below.Comments and suggestions by reviewers were valuable in clarifying key points of our manuscripts.We believe that the current version of the manuscript will meet the requirements of Life Science Alliance.: mkikkawa@m.u-tokyo.ac.jpAnswers to Reviewer #1's commentsReviewer #1 (Comments to the Authors (Required)): Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance.
Thank you for submitting your Research Article entitled "Calaxin is a key factor for calcium-dependent waveform control in zebrafish sperm".It is a pleasure to let you know that your manuscript is now accepted for publication in Life Science Alliance.Congratulations on this interesting work.